The physiological effects of cocaine are mediated by inhibition of dopamine reuptake, and subsequent enhancement of dopaminergic neurotransmission. We have recently made a surprising and unexpected finding that physiological effects of cocaine in animals require methylation of adenosine residues in mRNA. Adenosine methylation was discovered last year, and constitutes the first, and potentially only, reversible mRNA modification. Our group showed that methylation of adenosine residues in mRNA to form N6- methyladenosine (m6A) is highly prevalent and affects over 8,000 transcripts in the brain. Furthermore, adenosine methylation and demethylation is regulated by signaling pathways, suggesting that adenosine methylation, like protein phosphorylation, may be a fundamental mechanism mediating effects of signaling pathways in cells. The goal of this application is to substantially advance our understanding of this newly discovered and largely mysterious mRNA modification that appears to have a central role in synaptic transmission. As part of our overall goal to understand the cellular functions of m6A and to determine how it regulates dopaminergic neurotransmission, the specific aims of this proposal are: (1) To map m6A sites in the midbrain transcriptome at single-nucleotide resolution. We will develop a next-generation sequencing approach that incorporates a novel chemical biology strategy to label m6A in living cells. These experiments will provide new insights into the potential biological functions of m6A and will result in the identification of m6A sites in the midbrain transcriptome for analysis and mutagenesis in Aims 2 and 3; (2) To determine how FTO affects the translation of its target mRNAs. The mechanism by which mRNAs are translationally suppressed by m6A is not known. In this aim, we will address the potential molecular pathways by which m6A influence mRNA fate in cells. (3) To identify the FTO targets that is required for cocaine signaling in dopaminergic neurons. Here we will test FTO-target mRNAs that influence neurotransmission and synaptic signaling for their ability to rescue the impaired cocaine effects in FTO-/- slices. These experiments will elucidate the first signaling pathway linking methylation of specific mRNAs to physiological changes in neurons. The experiments proposed here will begin to decipher the role of a novel, highly prevalent, and medically important mRNA modification that is poised to have important roles in dopamine neurotransmission as well as other signaling pathways in neurons and other tissues.